Abstract
The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.
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Data availability
Datasets are deposited in the NCBI Gene Expression Omnibus using the following accession code: RNA-seq, GSE138459. Additional information can be found in the Nature Research Reporting Summary. Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author. Source data are provided with this paper.
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Acknowledgements
We thank members of the Thompson and Finley laboratories for discussion and critical feedback. S.A.V. is a Senior Fellow with the Parker Institute of Cancer Immunotherapy and is supported by a Burroughs Wellcome Fund Career Award for Medical Scientists. A.T.S. was supported by a Bridge Scholar Award from the Parker Institute for Cancer Immunotherapy and a Career Award for Medical Scientists from the Burroughs Wellcome Fund. This work was additionally supported by the Memorial Sloan Kettering Cancer Center Support Grant no. P30 CA008748 and R25 Training Grant no. AI140472-01A1.
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S.A.V. and C.B.T. conceived the study. S.A.V. performed all experiments with assistance from M.A.H. M.B. and J.R.C. assisted with LC–MS, extracellular flux and nutrient consumption experiments. D.K.W., B.K., A.T.S., H.Y.C. and K.E.Y. assisted with analysis of RNA-seq data. M.S., P.S.H. and M.R.M.v.d.B. assisted with CAR-T cell experiments. C.B.T. provided additional work in conception and study guidance. S.A.V. and C.B.T. wrote the manuscript.
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C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He is also a former member of the Board of Directors and a stockholder of Merck and Charles River Laboratories. S.A.V. has received honoraria from Agios Pharmaceuticals and Rheos Pharmaceuticals, is an advisor for Immunai and has consulted for ADC Therapeutics. A.T.S. and D.K.W. are scientific founders and equity holders of, and receive consulting fees from, Immunai. A.T.S. received funding support from 10x Genomics and Arsenal Biosciences. K.E.Y. is an advisor for Immunai. H.Y.C. is a co-founder of Accent Therapeutics and Boundless Bio and is a consultant for 10x Genomics, Arsenal Biosciences and Spring Discovery.
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Extended data
Extended Data Fig. 1 Chronic T cell stimulation induces T cell exhaustion.
All experimental analyses were conducted eight days after initial stimulation unless otherwise specified. a–c, Expression of inhibitory immunoreceptors (PD-1, LAG-3, PD-L1) and intracellular cytokine production (IFN-γ and TNF) in acutely and chronically stimulated T cells following re-stimulation with PMA and ionomycin. d, Expression of Glut1 in acutely or chronically stimulated OT-I T cells with or without restimulation using bead-bound anti-CD3. Actin is used as a loading control. Experiment was repeated three times with similar results. Uncropped blot can be found within Source Data. e, Gene set enrichment plot showing that genes associated with chronically stimulated polyclonal T cells in vitro are enriched for genes upregulated in exhausted CD8 + T cells (Texh) but not anergic T cells15. f, Killing of peptide-pulsed B16 cells. Luciferase-expressing B16 cells pulsed with Ova peptide at the indicated doses for 4 h were co-cultured with acutely or chronically stimulated T cells for 24 h. The following day, cells were lysed and luciferase expression was assessed using a luminometer. g, Normalized isotopologue abundance of intracellular lactate in acutely and chronically stimulated T cells following 6 h of re-stimulation by plate-bound anti-CD3 in the presence of U-13C-Glucose. Abundance was normalized to cell number at the time of harvest. h, Median lactate excreted per molecule of glucose consumed in acutely and chronically stimulated T cells following initial stimulation. P values were calculated by unpaired, two-sided Student’s t-test (f–h), relative to acutely stimulated T cells or based on 1,000 permutations by the GSEA algorithm and not adjusted for multiple comparisons (e). Data are presented as the mean ± s.d. of n = 3 biologically independent samples from a representative experiment. **P < 0.01.
Extended Data Fig. 2 Aerobic glycolysis is a hallmark of chronic stimulation-dependent terminal T cell dysfunction.
a, Extracellular acidification rate of acutely and chronically stimulated polyclonal T cells in media containing or lacking glucose as indicated. b, Extracellular acidification rate of acutely and chronically stimulated polyclonal T cells at baseline and in response to electron transport chain inhibition. c, Population doublings of acutely and chronically stimulated polyclonal CD8 + T cells following initial stimulation. d, Viability of acutely and chronically stimulated T cells as determined by forward scatter and DAPI exclusion. e, Intracellular TOX expression and proliferation as measured by dilution of Cell Trace Violet fluorescence of acutely or chronically stimulated T cells. f, Normalized expression of glycolytic genes in CD8 + T cell clusters from patients with basal and squamous cell carcinoma treated with immune checkpoint blockade19. g, Gene set enrichment plot showing that genes associated with terminally exhausted T cells isolated from murine B16 melanoma tumors8 are enriched for glycolytic genes. h, Correlation of glycolysis score (left) and TCA cycle score (right) with TCF7 expression in exhausted CD8+ T cell clusters from basal and squamous cell carcinoma patients treated with immune checkpoint inhibitors6. i, Gene set enrichment plot showing that chronically stimulated OT-I T cells in vitro significantly downregulate genes upregulated in progenitor Texh as compared to terminal Texh8. j, k, Intracellular cytokine production in acutely and chronically stimulated polyclonal T cells following re-stimulation. In (j), cells were cultured in the presence or absence of anti-PD-L1 (10 F.9G2) from D2-D8. In (k), “Chronic + 24 h rest” cells were rested in the absence of plate-bound anti-CD3 for 24 h prior to re-stimulation. Experiment was repeated three times with similar results. P values were calculated by unpaired, two-sided Student’s t-test (a–c) relative to acutely stimulated T cells or based on 1,000 permutations by the GSEA algorithm and not adjusted for multiple comparisons (f–i). Data are presented as the mean ± s.d. of n = 3 biologically independent samples from a representative experiment. ****P < 0.0001.
Extended Data Fig. 3 Chronic antigen stimulation impairs mitochondrial oxidation and ATP production.
a, Quantification of relative tricarboxylic acid cycle metabolite pool sizes in acutely and chronically stimulated T cells. Columns represent biological replicates for each condition. b, Oxygen consumption rate (OCR) of acutely or chronically stimulated T cells at baseline or in the presence of ATP synthase inhibition (Oligo), uncoupling agents (FCCP), inhibition of glucose uptake (2-DG), and complex III/IV inhibition (Rot/AA). c, Schematic depicting how oxidative metabolism of uniformly-labeled palmitate ([U-13C] palmitate) generates metabolites associated with the TCA cycle. Colored circles represent 13C-labeled carbons. d, Fractional labeling by [U-13C] palmitate of citrate, glutamate, fumarate, malate and aspartate in acutely and chronically stimulated T cells following re-stimulation. e, Proliferation of T cells acutely or chronically stimulated in the presence or absence of supplemental sodium acetate (5 mM), as measured by dilution of Cell Trace Violet fluorescence. f, Quantification of pool sizes of metabolite intermediates in nucleotide synthesis in acutely and chronically stimulated T cells. Heatmap depicts pool size relative to row median. Columns represent biological replicates for each condition. Experiment was repeated two times with similar results. P values were calculated by unpaired, two-sided Student’s t-test (a,b,d). Data are presented as the mean ± s.d. of n = 3 biologically independent samples from a representative experiment. **P < 0.01. ****P < 0.0001.
Extended Data Fig. 4 Oxidative stress limits T cell proliferative capacity.
a, Western blot depicting overexpression of FLAG-tagged recombinant NADH oxidase enzymes LbNOX and MitoLbNOX in T cells30. Experiment was repeated three times with similar results. Uncropped blot can be found within Source Data. b, Fluorescence intensity of acutely and chronically stimulated T cells expressing vector control, LbNOX, or MitoLbNOX following eight days in culture after loading with CM-H2DCFDA. c, Population doublings of acutely and chronically stimulated T cells expressing vector control, LbNOX, or MitoLbNOX. d, Fluorescence intensity of acutely and chronically stimulated T cells after loading with BODIPY-C11 to measure lipid peroxidation. Light-grey-shaded peak represents negative control. e, Fluorescence intensity of acutely or chronically stimulated T cells cultured with or without pharmacologic agents that impair ETC function following 2 days of initial stimulation. Cells were loaded with CM-H2DCFDA to measure ROS on D8 following initial stimulation. f, qRT-PCR of Myb and Tcf7 in acutely or chronically stimulated T cells with or without the addition of the indicated agents for 6 days following 2 days of primary stimulation. g–i, Expression of oxidative stress-related metabolic genes (“ROS score”) in tumor-infiltrating CD8 + T cells from basal and squamous cell carcinoma patients treated with immune checkpoint inhibitors19. In (g), ROS score in independent CD8 + T cell clusters is shown. In (h), ROS score in exhausted and memory T cell populations is shown according to clone size as measured by TCR sequencing; box center line=median, box limits=upper and lower quartiles, box whiskers=1.58 x interquartile range. In (i), correlation of ROS score with TCF7 expression in exhausted CD8 + T cells is shown. Only cells with non-zero TCF7 expression were included. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons post-test (g, i), or one-sided Student’s t-test relative to base mean (g, h). Data are presented as the mean ± s.d. of n = 3 biologically independent samples from a representative experiment. **P < 0.01. ***P < 0.001. ****P < 0.0001.
Extended Data Fig. 5 Endogenous anti-oxidant production is limiting for T cell proliferation.
a, Motif analysis of sites with increased accessibility in tumor-infiltrating CD8 + T cells (L7) as compared to T cells from Listeria-infected mice (E7) showing NFATc1 as among the motifs whose accessibility was most preferentially increased in L7 cells15. b, Intracellular calcium flux as measured by ratio of bound to unbound Indo-1-AM in acutely and chronically stimulated T cells, at baseline, in response to monomeric anti-CD3, and in response to receptor clustering (streptavidin). c, Gene set enrichment plot showing that chronically stimulated OT-I T cells are enriched for NFAT target genes. d, Expression of NFAT target genes (“nfat score”) in independent CD8 + T cell clusters. e, Correlation of expression of NFAT target genes (“nfat score”) with expression of oxidative stress-related metabolic genes (“ROS score”) in tumor-infiltrating CD8 + T cells from melanoma patients treated with immune checkpoint inhibitors. f, Fluorescence intensity of acutely and chronically stimulated T cells cultured with or without βME supplementation after loading with CM-H2DCFDA to measure ROS. Light-grey-shaded peak represents negative control. g, Proliferation of T cells acutely stimulated in the presence or absence of BSO or diamide as measured by dilution of Cell Trace Violet fluorescence. h, Expression of TCF-1 and TOX in chronically stimulated T cells cultured in the presence or absence of BSO. P values were calculated by one-sided Student’s t-test relative to base mean (d, e). ****P < 0.0001.
Extended Data Fig. 6 N-acetylcysteine reverses oxidative stress in chronically stimulated T cells.
a, Quantification of relative metabolite pool sizes as measured by LC-MS in chronically stimulated T cells cultured with or without N-AC. Colored dots represent intermediates in glutathione synthesis as indicated. Dashed lines represent cutoffs of p < 0.01 and log2 fold change > 0.5. b, ATP production by acutely or chronically stimulated T cells cultured with or without N-AC. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons post-test compared to acutely stimulated T cells (b). Data are presented as the mean ± s.d. of n = 4 biologically independent samples from a representative experiment (b). *P < 0.05.
Extended Data Fig. 7 Antioxidants restore T cell self-renewal during chronic stimulation.
a, Population doublings of chronically stimulated T cells with or without N-AC supplementation under normoxic (left) or hypoxic (right) conditions. Experiment was repeated two times with similar results. b, qRT-PCR of Tcf, Myb, and Prdm1 in acutely or chronically stimulated T cells with or without the addition of N-AC as indicated. c, Intracellular calcium flux as measured by ratio of bound to unbound Indo-1-AM in acutely and chronically stimulated T cells cultured with or without N-AC. d, Gene set enrichment plot showing that the addition of N-AC during chronic stimulation reduces expression of NFAT target genes. P values were calculated by unpaired, two-sided Student’s t-test relative to cells cultured without N-AC (a), one-way ANOVA with Sidak’s multiple comparisons post-test (b) or based on 1,000 permutations by the GSEA algorithm and not adjusted for multiple comparisons (d). Data are presented as the mean ± s.d. of n = 3 biologically independent samples from a representative experiment. *P < 0.05. **P < 0.01. ***P < 0.0001.
Extended Data Fig. 8 Antioxidants reverse endogenous tumor-associated T cell dysfunction.
a, Production of IFN-γ and TNF following re-stimulation with PMA and ionomycin in chronically stimulated T cells with or without N-AC supplementation under normoxic or hypoxic conditions. Experiment was repeated two times with similar results. b, c, Oxygen consumption rate (OCR) of OT-I T cells chronically co-cultured with B16 melanoma cells with or without anti-PD-L1 antibodies and with or without N-AC supplementation at baseline or in the presence of ATP synthase inhibition (Oligo), uncoupling agents (FCCP), or complex III/IV inhibition (Rot/AA). d, Production of IFN-γ and TNF following re-stimulation with PMA and ionomycin in chronically stimulated T cells with or without MitoTEMPO or Trolox supplementation as indicated. Experiment was repeated two times with similar results. P values were calculated by unpaired, two-sided Student’s t-test (c). Data are presented as the mean ± s.d. of n = 4 biologically independent samples from a representative experiment (b, c). *P < 0.05.
Extended Data Fig. 9 Gating strategy for fluorescence activated cell sorting analysis.
For both polyclonal and OT-I transgenic T cells, gating was perform as shown. First, doublet exclusion was performed on cells gated by FSC-H versus FSC-W. Then, doublet exclusion was performed on cells gated by SSC-H versus SSC-W. Viable cells were identified by FSC-A and Live/Dead Blue exclusion. Finally, CD8 positivity was assessed by fluorescence in the BV-786 channel.
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Source Data Fig. 3
Images depict uncropped images for western blots in Fig. 3b. Red boxes contain cropped area shown in main figure.
Source Data Extended Data Fig. 1
Images depict uncropped images for western blots in Extended Data Fig. 1d. Red boxes contain cropped area shown in main figure.
Source Data Extended Data Fig. 4
Images depict uncropped images for western blots in Extended Data Fig. 4a. Red boxes contain cropped area shown in main figure.
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Vardhana, S.A., Hwee, M.A., Berisa, M. et al. Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nat Immunol 21, 1022–1033 (2020). https://doi.org/10.1038/s41590-020-0725-2
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DOI: https://doi.org/10.1038/s41590-020-0725-2
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